Local anesthesia is generally considered to involve specific binding to receptors, most probably to the sodium channel whose gate is opened. Against the receptor theories, nonspecific membrane perturbation, represented by the membrane stabilization concept, has also been proposed. The fact that part of the aromatic amine local anesthetics exists as a positively charged form is consistent with receptor binding because biological membranes usually carry negative surface charges. Nevertheless, atypical local anesthetics, such as alcohols, tranquilizers, antihistaminics, barbiturates, narcotics, general anesthetics, etc., also block nerve conduction. The diversity of drugs is difficult to reconcile with the idea that receptor binding is the sole mechanism of local anesthesia. Presumably, local anesthesia is caused by multiple mechanisms. Coexistence of charged and uncharged species of local anesthetics at a physiological condition has been a matter of concern as to which is the biologically active species. Despite the importance of accurate dissociation constants to differentiate between the actions of the two species, systematic effort to determine this parameter has been few. In the last funding period, we used the nonlogarithmic linear titration method to measure accurate dissociation constants. The effort will continue. The main objective of the present project, however, is to evaluate the interaction of charged local anesthetics with membranes and macromolecules. Membrane perturbation studies so far reported are concerned predominantly with uncharged species. With the availability of our data on dissociation constants, charged molecule amounts can be estimated with reasonable accuracy. Studies on the interaction with lipid monolayers, vesicle membranes, planar lipid bilayers, and macromolecules will be continued. The present project will also include studies of anesthetic effects upon liquid membranes with mobile ion carriers. The experimental methods consist of the standard surface and colloid chemistry and were used successfully during the previous funding period.